Project Abstract This application is being submitted in response to NOT-AI-19-040. The parent grant aims to use expanded recipient Tregs to enhance the level and duration of chimerism achieved in monkeys receiving minimal, non- myeloablative conditioning and allogeneic bone marrow transplantation to induce robust allograft tolerance. Based on strong results in a mouse-to-mouse allotransplantation model, co-transplantation of recipient-derived polyclonal Tregs is a promising approach to achieving this goal. Studies in the non-human primate (NHP) model are essential for the optimal translation of this approach to the clinic. Our efforts to apply the Treg approach to NHPs have led to improvements in chimerism and tolerance, but we have not yet achieved the permanent chimerism seen in the mouse model. We have explored several polyclonal Treg expansion protocols and antigen-presenting cell sources, including artificial APCs and activated B cells from multiple donors. While additional variations warrant testing, considerable time and expense is required to test each one in NHPs. A more nimble model that mirrors the human and monkey in the challenge of achieving durable mixed chimerism would help to guide the NHP studies. Humanized immune system (HIS) mice generated by transferring human fetal liver CD34+ cells to immunodeficient mice that receive human fetal thymus tissue under the kidney capsule provide such a model. The robust human immune systems generated in these mice permit only transient chimerism to be achieved when allogeneic human CD34+ cells are administered following conditioning with a regimen based on the one used in the monkey model and related clinical trials. Normal Tregs are generated and can be expanded in vitro. Therefore, this HIS mouse model (termed HU/HU thymus/HSC mice) provides an excellent platform for testing the ability of expanded recipient Tregs to promote durable mixed allogeneic chimerism induction. Due to concerns about the availability and use of human fetal tissue in this model, we aim to modify it to avoid using human fetal tissue, meeting the requirement for NOT-AI-19-040. Specifically, we aim to: 1) Generate HIS mice with pig fetal thymus and human cord blood CD34+ cells and establish baseline Treg development and expansion parameters as well as capacity to promote mixed allogeneic chimerism; 2) Generate HIS mice with cord blood CD34+ cells and pig ?hybrid? thymus containing human pluripotent stem cell- derived thymic epithelial cells (hPSC-TECs); determine the impact of hPSC-TECs on positive selection of HLA- restricted TCRs and Treg development. Adaptations in Treg use resulting from the HIS mouse model of mixed chimerism induction will be directly applied to the NHP model in the parent grant and ultimately in clinical trials.